Journal of Chemical Ecology, Vol. 16, No. 4, 1990

CHEMOSENSORY RESPONSES OF COWPEA WEEVIL, Callosobruchus chinensis TO AN AQUATIC WEED,

WATER HYACINTH, Eichhornia crassipes (MART) SOLMS

U S H A R A N I and K A I S E R J A M I L

Entomology Division, Regional Research Laboratory Hyderabad 500 007, Andhra Pradesh, India

(Received February 27, 1989; accepted July 10, 1989)

Abstract--The behavioral responses of cowpea weevil, Callosobruchus chi- nensis to the leaf extracts of the aquatic weed, water hyacinth, Eichhornia crassipes (Mart) Solms. was evaluated by three different methods. The petro- leum ether-soluble fraction of E. crassipes induced high weevil attraction in all methods employed. The olfactometer assay provided a reading on weevil chemosensory responses, whereas the free-choice tests measured response and preference of weevils to the untreated or extract-treated food grain of their choice over a longer period. The extracts were also presented under no- choice test conditions to assay them for the presence of oviposition stimu- lants. In all these methods the extracts of water hyacinth evoked a quick directional response. Another interesting feature is that the extract, besides producing attraction, also caused mortality of the insects.

Key Words--Behavioral responses, cowpea weevil, Callosobruchus chinen- sis, Coleoptera, Curculionidae, water hyacinth, Eichhornia crassipes, olfac- tometer.

INTRODUCTION

Al though chemosenso ry react ions have long been recogn ized as essent ial per-

cep t ive processes in Co leop te ra associa ted wi th s tored grain, only recent ly have

a few studies e x a m i n e d this aspect o f s tored-product insects (Gomez et a l . ,

1983). Whi l e a t tempt ing to eva lua te the a l tomonic pr inciples o f Parthenium

hysterophorus, Sharma and Tosh i (1977) found that the ace tone and methanol

extracts o f this w e e d were a t t ract ive to the red flour beet le , Triboleum casta-

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0098-0331/90/0400-1269506.00/0 �9 1990 Plenum Publishing Corporation

1270 RANI AND JAMIL

neum. Olfactory responses of rice weevil, Sitophilus oryzae to fractions of rice grains, and Trogoderma granarium to odors of wheat, feces, and fecal com- ponents were also reported (Honda et al., 1969; Stanic and Shulov, 1969). However, reports on the plant products affecting the chemosensory behavior of stored product pests are very scanty. In particular, the weeds and wild plants that are of no direct use for humans can be exploited for the protection of stored commodities from pest insects. A few such plants already have been reported to be useful for this purpose. The insecticidal value of the essential oil of Acorus calamus has been examined against the pulse beetle, Bruchus chinensis (Yadava, 1971). The oil protected the grains for many months from the attack of beetles. The efficacy of two wild plants, Hyptis spicigera and Cassia nigricans, in con- trolling the bean weevil, AcanthosceIides obtectus, was determined by Lambert et al. in 1985.

Water hyacinth, Eichhornia crassipes (Mart) Solms. is a floating aquatic weed of South American origin that has become a major problem in India. Dense mats of water hyacinth frequently pose problems in the commercial and recreational use of water, increase damage resulting from flooding, and cause major changes in the ecology of infested areas. Various control measures and utilization of this weed have been investigated extensively (Gopal and Sharma, 1981). Insecticidal and growth-regulatory properties of water hyacinth against a few storage pests are already reported (Rani and Jamil, 1989). In this inves- tigation behavioral assay of chemical extracts of water hyacinth was undertaken to establish the presence of attractant and/or repellent properties that affect the behavior of the cowpea weevil. The bruchid beetles have obvious economic importance, because they breed on legumes and consume valuable proteins that would otherwise be eaten by man, reduce seed quality, and lower seed germi- nation.

METHODS AND MATERIALS

Water hyacinth plants with long petioles were collected from a local pond near the laboratory. Leaves of the plants were separated and air-dried for three days. The dried leaves were coarsely ground by using a mortar and pestle. The ground material (500 g) was extracted with petroleum ether (bp 40-60~ in a Soxhlet apparatus. The resulting extract was concentrated under reduced pres- sure to remove the solvent.

A stock solution containing 100 mg/ml of the extract in acetone was pre- pared and refrigerated when not in use. Lower concentrations of 80, 60, 40, and 20 mg/ml were obtained by dilution of the stock solution with the solvent. Only these dosages were used in all the departments.

Collosobruchus chinensis adults were obtained from laboratory cultures

CHEMOSENSORY RESPONSES OF COWPEA WEEVIL 1271

maintained on green grain, Vigna radiata, which also were used in all the exper- iments described below. The grains were cleaned and later disinfested by keep- ing at 60~ for a 3-hr period. These samples were brought to equilibrium prior to use for experimental purpose. All cultures and experiments were conducted in a controlled temperature and humidity (CTH) room at 27 _+ 1~ and 65- 75 % relative humidity using culturing procedures previously described by Dick and Credland (1984).

Beetles used in the tests were separated daily from the green grain culture media. Newly emerged weevils were selected because weevils younger than 4 days of age were more sensitive to odors than older ones (Honda et al., 1969). Also, possible sex pheromone effects would be minimized. According to Sharma & Deora (1980), sex pheromone may be produced and released by weevils 5 days old. The absence of pheromone effects was confirmed by two tests: (1) when 25 newly emerged females were caged in the sample tube, no attractant response was elicited from the unsexed adults in the stem of the Y tube; and (2) when 25 unsexed adults were caged in the sample tube, no aggregation response was elicited from the unsexed adults in the stem. These tests were replicated five times.

Olfactometer Assay. Attractancy and/or repellency of the extract for C. chinensis was assayed by studying the behavior and response of the weevils exposed to the compound. The apparatus used to study the olfactory behavior of the weevils is a Y-type olfactometer. The lengths of the stem and two arms of the Y tube are 20 cm and the diameter is 1 cm. Smaller tubes of 5 cm length and 1 cm diameter, which could fit snugly inside the ends of both the arms, were used for placing cotton swabs, each weighing 30 mg, impregnated with 1 ml of sample (test) or acetone solvent (control). The solvent was evaporated by air-drying. The test and control cotton swabs placed in the smaller tubes were inserted at the open ends of the bigger tube. Twenty 1-day-old unsexed adult beetles were introduced into the olfactometer through the free end of the stem of the Y tube after a priming period of 30 rain, during which the odor concen- tration built inside the two arms.

The distribution of 20 experimental insects in the control (odor-free) and test (odor-containing) sectors of the tube was recorded every 10 min for 2 hr. At least five such experiments were conducted, and the number of insects aggre- gating in the test and control sectors was averaged for all readings. Attraction and/or repulsion was determined by using the formula [T - C/T + C] • 100 where T is the number of insects in the test and C in the control sectors. Index of attractancy above 50 indicated attraction; below 50 indicated repulsion.

Free-Choice Tests. To measure the preference of the weevils for treated and untreated areas, free-choice tests were conducted. The olfactometer was of the alternative chambers type and featured a glass trough (15 cm diam.). Three small petri plates (5 cm diam.) were cemented equidistantly around the wall,

1272 Rayx AND JAMIL

and in each was mixed 32.5 g of green grain with various doses of the extract, 80, 60, 40, and 20 mg dissolved in 2 ml of acetone separately and after the solvent evaporated under an airstream. Grains treated with acetone alone served as controls. Twenty 1-day-old unsexed weevils were introduced in the center of the apparatus which was then closed with a thick cloth and elastic band. The experimental set up was kept in a temperature-controlled incubator (28 + 1 ~ that also provided darkness. The number of weevils found in each Petri plate was recorded 15, 30, and 60 min after introduction.

No-Choice Test. An experiment was conducted to test the effects of pro- longed exposure to water hyacinth extract on C. ehinensis. In this treatment, 20 weevils were placed individually into 50-ml plastic cups. The cups were filled with 10 g of grain sprayed and air-dried with 2 ml of the extract, at various concentrations. The cups were then capped with a thick cloth held in position with a rubber band. Twenty additional weevils placed in identical cups con- taining the grains with 20 ml of acetone, served as a control. Both treatments were held at 28 +__ 1~ for 24 hr to compare adult mortality. For each dose, five replicates were used.

RESULTS AND DISCUSSIONS

The petroleum ether extract of powdered dry leaves (500 g) of water hyacinth yielded 4.72 g of a dark green thick liquid. This extract was reported to be toxic to cowpea weevils in contact as well as in food grain treatment methods (Rani and Jamil, 1989). In the present investigation, three types of behaviorial responses were tested. The olfactometer assay provided a reading on weevil chemosensory response to extracts during a 3-hr period. The free- choice tests measured response effects by permitting weevils to be attracted to or feed on extract-treated food grains of their choice over a longer period. Lastly, these same extracts were presented under no-choice test conditions to assay them for the presence of oviposition stimulants.

The petroleum ether-soluble fraction of E. crassipes induced high weevil attraction in most of the methods employed. In the olfactometer assay, the extract showed a high range of attraction to pulse beetles. Fifteen minutes after release of the weevil, the attraction index was significantly higher than the index value of the control. The response was more clear-cut 30 and 45 min after release. Within 10 min of their introduction into the olfactometer, insects started moving toward the treated arm. All aggregated on the cotton swab. After a period of 45 rain, the insects died on the extract-impregnated cotton (Table 1). The results obtained at all concentrations tested were similar.

When weevils were allowed to crawl on grains containing extract, there

CHEMOSENSORY RESPONSES OF COWPEA WEEVIL i273

TABLE 1. RESPONSE OF PULSE BEETLES TO PETROLEUM ETHER EXTRACT OF WATER HYACINTH LEAF IN OLFACTOMETER

Number of insects found oll a

Time Treated Control Index of Mortality Dosage (min) sector sector attractancy (%)

80 10 1.0 0 100.0 Nil 15 8.0 2.8 48.20 Nil 30 13.2 2.4 69.20 10.0 45 20.0 0 100.00 100.0

60 10 2.0 0 100.00 Nil 15 8.0 2.5 52.40 Nil 30 10.8 2.2 66.20 68.0 45 20.0 0 100.00 100.0

40 10 1.0 0 i00.00 Nil 15 10.0 3.0 53.80 Nil 30 15.2 2.1 75.70 Nil 45 19.3 0 100.00 Nil

20 10 1.2 0 100.00 Nil 15 4.0 3.2 11.11 Nil 30 12.0 3.2 57.90 Nil 45 17.4 1.8 81.20 Nil

a Mean of five replicates (20 insects per replicate).

was a well-defined reaction. The weevils were attracted in large numbers by treated grain and after a period of 1 hr all of them were found dead. This

occurred in all the concentrations tested except the lower dilution, i.e., 20 rag/ 2 ml; at this dilution insects did not die but became sluggish.

In no-choice tests, the extract produced 100% mortality of the weevils at

80 and 60 mg/2 ml, and a lesser dose of 40 mg/2 ml caused 40% mortality of

the insects within 24 hr. However, remaining insects were also found dead after

a period of 72 hr. No lethal effect was noted at the dose of 20 mg/2 ml even after 72 hr of treatment (Table 2).

The leaf extract of water hyacinth contained compounds that evoked a quick

directional response and also an insecticidal response. It is possible that the extract contains more than one chemical, one that serves as a stimulant on both

sexes, and another that serves as a toxicant. Further elucidation of the questions concerning the identity of the com-

pound(s) involved in the behavioral responses of the rice weevil awaits further fractionation of the petroleum ether extracts and characterization of these frac-

1274 RANI AND JAMIL

TABLE 2. RESPONSES OF PULSE BEETLE TO WATER HYACINTH EXTRACT IN NO-CHOICE TEST

Mortality (%) after

Dosage (/~g/ml) 24 hr 72 hr

80 100 100 60 100 100 40 40 100 20 0 0

tions chemically and behaviorally. Goswami et al. (1983) reported a significant

amount of stigmasterol (0.07% dry weight) in water hyacinth plants and the

possibility of its enrichment 10-fold by anaerobic digestion of the plant mate-

rial. Most of the sterols were present in the free form, which is significant in

considering water hyacinth as a source of sterol. These findings were further

confirmed by Jamil et al. (1988). Preliminary chemical analysis of the extract that caused molting abnormalities in Corcyra cephalonica revealed the presence

of sterols.

The combined contact toxicity and attraction reported here make E. cras-

sipes a good candidate for further development as a short-term grain protectant

against infestation by cowpea weevils.

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CHEMOSENSORY RESPONSES OF COWPEA WEEVIL 1275

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